A central challenge in the field of motor control is to understand how descending systems control spinal cord interneurons (INs). However, descending axons target thousands of spinal INs and form complex neural circuits. This complexity limits the usefulness and efficiency of conventional investigation techniques. To solve this roadblock and accelerate progress, we recently developed alternative approaches that transfer functional connectivity studies to the in vitro mouse. We use optical imaging to record individual and population neuronal activity following stimulation of subcortical descending systems. This novel approach allows us to study synaptic connections in descending networks with an efficiency and throughput that has not been possible previously in mammals. In this proposal, we focus on a major player in motor coordination, the reticulospinal (RS) descending system. We propose to identify the functional connections between RS neurons and a population of commissural INs with descending axonal projections called dCINs. CINs are strategically interposed between RS input and motor output to play an important role in locomotor rhythm generation and interlimb coordination. However, they are heterogeneous with respect to transmitter phenotype, input-output connectivity and functional role during movement. We have begun to elucidate organizational principles of RS-dCIN system and made two important preliminary findings. We found; (i) two distinct groups of medullary RS neurons that differentially activate axial and hindlimb motoneurons, and (ii) three subpopulations of dCINs that respond to either RS group or to both. Thus, RS neurons are organized in discrete groups that recruit dCINs selectively. Here, in Aim 1, we propose to test two forms of selectivity that RS neurons may use to recruit dCINs. First, we will test segmental selectivity by taking advantage of the known spinal segmental differences in composition of axial, flexor and extensor motoneurons. Then, we will test transmitter phenotype selectivity using transgenic approaches. In Aims 2 and 3, we will determine the extent to which RS-dCIN recruitment depends on the excitability and behavioral state of the spinal motor network. Successful completion of these aims will significantly advance our understanding of the functional organization of the RS system both during quiescence and during the active engagement of a complex motor behavior. Our long-term goal is to define organizational principles by which brainstem descending systems control motor output via spinal interneurons. Such knowledge will undoubtedly provide greater insight into circuit dysfunction after injury and aid identify rational strategies for moto recovery.